The full induction of human apoprotein A-I gene expression by the experimental nephrotic syndrome in transgenic mice depends on cis-acting elements in the proximal 256 base-pair promoter region and the trans-acting factor early growth response factor 1

To identify molecular factors regulating apo A-I production in vivo, we induced in transgenic mice the experimental nephrotic syndrome, which results in elevated levels of HDL cholesterol (HDL-C), plasma apo A-I, and hepatic apo A-I mRNA. Human (h) apo A-I transgenic mice with different length 5' flanking sequences (5.5 or 0.256 kb, the core promoter for hepatic-specific basal expression) were injected with nephrotoxic (NTS) or control serum. With nephrosis, there were comparable (greater than twofold) increases in both lines of HDL-C, h-apo A-I, and hepatic h-apo A-I mRNA, suggesting that cis-acting elements regulating induced apo A-I gene expression were within its core promoter. Hepatic nuclear extracts from control and nephrotic mice footprinted the core promoter similarly, implying that the same elements regulated basal and induced expression. Hepatic mRNA levels for hepatocyte nuclear factor (HNF) 4 and early growth response factor (EGR) 1, trans-acting factors that bind to the core promoter, were measured: HNF4 mRNA was not affected, but that of EGR-1 was elevated approximately fivefold in the nephrotic group. EGR-1 knockout (EGR1-KO) mice or mice expressing EGR-1 were injected with either NTS or control serum. Levels of HDL-C, apo A-I, and hepatic apo A-I mRNA were lowest in nonnephrotic EGR1-KO mice and highest in nephrotic mice expressing EGR-1. Although in EGR1-KO mice HDL-C, apo A-I, and apo A-I mRNA levels also increased after NTS injection, they were approximately half of those in the nephrotic EGR-1-expressing mice. We conclude that in this model, basal and induced apo A-I gene expression in vivo are regulated by the trans-acting factor EGR-1 and require the same cis-acting elements in the core promoter.     

Identification of cis-acting elements in the SUC2 promoter of Saccharomyces cerevisiae required for activation of transcription

We analyzed the effects of site-directed mutations in the SUC2 promoter of Saccharomyces cerevisiae. Analyses were performed in wild-type as well as mig1 and tup1 mutant strains after the promoter mutants were reintroduced into the native SUC2 locus on the left arm of chromosome IX. Mutation of the two GC boxes revealed that these elements play two distinct roles: they are, as expected, required for Mig1-mediated repression but they are also necessary for activation of the SUC2 promoter in response to glucose limitation. The individual GC boxes are functionally redundant with regard to Mig1-mediated repression, however, only the upstream GC box is essential for high level expression of SUC2. Microccocal nuclease sensitivity of the SUC2 promoter in derepressed cells was reduced in the GC box mutant promoters, particularly in the vicinity of the TATA box. The difference in nuclease sensitivity between wild-type and GC box mutant promoters was not evident in tup1- cells. The formation of nuclease-resistant chromatin does not require the GC boxes, indicating that other cis-acting elements can serve to recruit the Ssn6-Tup1 co-repressor complex to the SUC2 promoter.     

Analysis of the human ferrochelatase promoter in transgenic mice

Ferrochelatase catalyzes the chelation of ferrous iron and protoporphyrin to form heme. It is expressed as a housekeeping gene in all cells, but is upregulated during erythropoiesis. Ferrochelatase activity is deficient in the inherited disease protoporphyria as a result of heterogeneous mutations. Although human ferrochelatase is transcribed from a single promoter in both nonerythroid and erythroid cells, previous studies using transient transfection assays failed to demonstrate erythroid-specific increased expression from 4.0 kb of the human ferrochelatase promoter containing the erythroid cis-elements, GATA and NF-E2. The present study analyzes the in vivo regulation of the ferrochelatase gene to provide insights into the mechanism of its erythroid-specific enhancement. Transgenic (TG) mouse lines were generated in which the luciferase reporter gene was driven by either a 150-bp ferrochelatase minimal promoter (-0.15 TG) or by a 4.0 kb extended 5' upstream region (-4.0 TG). Expression of the -4.0 TG transgene was generally consistent with the endogenous gene during embryonic development and in nonerythroid and erythroid tissues as demonstrated by Northern blotting and mRNA in situ hybridization. The -4.0 TG was expressed at a higher level than the -0.15 TG in nonerythroid and erythroid tissues, including during extramedullary erythropoiesis induced by n-acetylphenylhydrazine injection. The enhanced erythroid expression of the -4.0 TG correlates with the appearance of a DNase I hypersensitive site in the 5' flanking region of the transgene. Therefore, in the context of chromosomal integration, the 5' flanking region of the ferrochelatase gene is necessary and sufficient to confer high levels of transgene expression in erythroid tissue.     

Constitutive expression of terminal deoxynucleotidyl transferase in transgenic mice is sufficient for N region diversity to occur at any Ig locus throughout B cell differentiation

N region diversity in Ag receptors is a developmentally regulated process in B and T cells that correlates with the differential expression of terminal deoxynucleotidyl transferase (TdT). Absent in fetal and newborn mice, TdT expression is restricted to early T and pro-B cells in adults. To extend the TdT expression pattern throughout B cell ontogenesis, we generated transgenic mice carrying a TdT cDNA under the regulatory elements of the N-myc gene and the IgH enhancer. High expression was observed in secondary lymphoid organs consistent with TdT activity beyond the pre-B cell stage. This suggests that TdT transgene expression is not down-regulated as is the endogenous gene. Unlike normal mice, extensive N region diversity was found in rearranged lambda light chain genes of adult transgenic animals. Therefore, expression of TdT appears sufficient for N region diversity to occur at any Ig locus. More importantly, expression of the transgene takes place during fetal development. As a consequence, the potential fetal B cell repertoire is modified as both rearranged heavy and light chain genes now show N region additions. Constitutive expression of TdT throughout B cell differentiation does not therefore appear deleterious and suggests that TdT is recruited only to participate in the V(D)J recombination process.     

Ocular abnormalities in transgenic mice harboring mutations in the type II collagen gene

PURPOSE: To characterize the morphological changes in the eyes of transgenic mice harboring different mutations in type II collagen gene to elucidate the function of this collagen in the eye, and to find out whether these animals could function as models for the human arthro-ophthalmopathies of the Kniest, Stickler and Wagner types. METHODS: Three genetically engineered mouse lines representing two types of mutations in the triple-helical domain of type II collagen and their nontransgenic littermates used as controls were analyzed on day 18.5 embryonic development. After genotyping by polymerase chain reaction (PCR) and Southern hybridization the embryos were prepared for routine histology. Polarization microscopy was done on hyaluronidase-treated sections. RESULTS: Histological analysis revealed several genotype-dependent abnormalities in the eyes of the transgenic mice. Most striking changes were observed in the vitreous architecture; in one line of mice the vitreous was tightly packed in the posterior region of the vitreous space with thick fibrils, empty cavities and dense membrane-like material. The other mutation resulted in reduced filament density of the vitreous. In the most severely affected phenotype the internal limiting membrane was detached from the retinal layers and was markedly thickened, and the posterior lens capsule was thickened. The anterior chamber was shallow or absent in all transgenic lines but was well formed in the normal animals. Changes were also observed in the lens, corneal and scleral structures. CONCLUSIONS: The ocular changes observed in transgenic mice harboring mutations in type II collagen gene show similarities to the human ocular findings in Kniest dysplasia, and in Stickler and Wagner syndromes. We therefore propose that these animals could serve as models for systematic analysis of vitreoretinal degeneration and other abnormalities, as seen in these syndromes.     

Brain cell type specificity and gliosis-induced activation of the human cytomegalovirus immediate-early promoter in transgenic mice

Human cytomegalovirus (HCMV) can cause debilitating, sometimes fatal, opportunistic infections in congenitally infected infants and in immunodeficient individuals such as patients with the acquired immunodeficiency syndrome (AIDS). Molecular mechanisms that determine cell type specificity of HCMV infection and latency are poorly understood. We recently described a transgenic mouse model for analysis of HCMV major immediate-early (IE) promoter regulation and showed that sites of IE promoter activity during murine embryogenesis correlate with known target tissues of congenital HCMV infection in human fetuses (Koedood et al., 1995). Among various permissive human tissues, the brain is a site where HCMV infections can be devastating. Here, we have used immunohistochemical double-labeling analysis to identify specific cell types with HCMV-IE promoter activity in brains of transgenic mice at several postnatal stages. IE promoter activity was restricted to some endothelial cells, ependymal cells, choroid plexus epithelia, and neurons at discrete locations in the forebrain, brainstem, and cerebellum. Endothelial cells and neurons with activity were proportionately more abundant in neonatal than in adult brains. Although the IE promoter was normally silent in most astrocytes, activity was strongly induced in reactive astrocytes in response to a neocortical stab lesion. The findings support a model, consistent with clinical literature on HCMV encephalitis, whereby tissue damage and gliosis caused by HCMV infection of endothelial and ependymal cells progressively renders adjacent permissive neurons and reactive astrocytes accessible to infection. This transgenic model system should facilitate identification of factors that regulate the HCMV IE promoter with regard to infection permissivity and reactivation from latency.     

Adenovirus E1A expression controlled by the red pigment promoter in transgenic mice: a model for developmental abnormalities of the eye

Four transgenic founder mice were produced that carry the adenovirus E1A gene under control of the human red pigment promoter. Two of the founder animals passed the transgene to progeny, and one line was bred to homozygosity. The line of mice homozygous for the transgene expressed E1A-specific RNA at a low level in their eyes; no expression could be detected in other tissues that were tested. Either the founder animals or their progeny exhibited developmental abnormalities of their eyes, including a small eye phenotype, retinal dysplasia, anterior cleavage syndrome, and retinal pigment epithelium dysplasia. Individuals from the same line of mice exhibited subsets of the abnormalities, e.g., many animals had one normal size eye and one small eye, even though both eyes contained E1A-specific RNA. No tumors resulted from E1A expression in animals monitored for 22 months.     

Evaluation of the combined assay of alpha-feto protein and gamma-glutamyl transpeptidase isoenzyme II in the diagnosis of primary hepatic carcinoma

Serum alpha-feto protein (AFP) and gamma-glutamyl transpeptidase isoenzyme II (gamma-GTP-II) were assayed in 104 patients with primary hepatic carcinoma (PHC), gamma-GTP-II had a higher specificity of 95.7%, while AFP a higher sensitivity of 84.6%. There was no significant difference in the positive diagnostic rate of the two markers in patients with AFP > or = 400 micrograms/L and with AFP < 400 micrograms/L. Nor was there any difference if the patients were divided into four groups according to the serum AFP levels of 200 micrograms/L, 400 micrograms/L and 1,000 micrograms/L. Altogether 97 of the 104 PHC patients were detected when the two markers were assayed in combination (40 out of 42 PHC patients with AFP > or = 400 micrograms/L and 57 out of 62 PHC patients with AFP < 400 micrograms/L). These results suggest that combined assay of the two serum PHC markers will increase the detecting rate of PHC and be of value especially in those patients with normal or low serum level of AFP.     

The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice

The human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) is one of the first promoters to activate upon infection. To examine HCMV MIEP tissue-specific expression, transgenic mice were established containing the lacZ gene regulated by the MIEP (nucleotides -670 to +54). In the transgenic mice, lacZ expression was demonstrated in 19 of 29 tissues tested by histochemical and immunochemical analyses. These tissues included brain, eye, spinal cord, esophagus, stomach, pancreas, kidney, bladder, testis, ovary, spleen, salivary gland, thymus, bone marrow, skin, cartilage, and cardiac, striated and smooth muscles. Although expression was observed in multiple organs, promoter activity was restricted to specific cell types. The cell types which demonstrated HCMV MIEP expression included retinal cells of the eye, ductile cells of the salivary gland, exocrine cells of the pancreas, mucosal cells of the stomach and intestine, neuronal cells of the brain, muscle fibers, thecal cells of the corpus luteum, and Leydig and sperm cells of the testis. These observations indicate that the HCMV MIEP is not a pan-specific promoter and that the majority of expressing tissues correlate with tissues naturally infected by the virus in the human host.     

cis-acting elements that mediate the negative regulation of Moloney murine leukemia virus in mouse early embryos

We have addressed the question of the nature of Moloney murine leukemia virus (MoMuLV) repression in mouse embryos by assaying for the transient expression of MoMuLV-derived constructs microinjected into early cleavage embryos. We show that the same cis-acting DNA sequences responsible for the block in MoMuLV expression in embryonal carcinoma cell lines operate in early embryos: (i) the MoMuLV long terminal repeat is nonfunctional, and (ii) the +147 to +163 repressor binding site, or negative regulatory element, negatively regulates the expression from an active promoter.     

Expression of the Zinnia TED3 promoter in developing tracheary elements of transgenic Arabidopsis

To determine the regulatory mechanism of gene expression in the early stages of tracheary element (TE) differentiation, we isolated and characterized a genomic fragment of TED3 whose mRNA is expressed preferentially in differentiating TEs 12-24 h before morphological changes in the in vitro Zinnia system. Transgenic Arabidopsis plants with a chimeric gene of the 537 bp TED3 promoter and the beta-glucuronidase (GUS) reporter gene indicated the strong expression of the GUS gene by the TED3 promoter in TEs, in particular in immature TEs as well as stipules and trichomes. GUS expression driven by the promoter was also induced in callus, in which GUS activity was localized in immature TEs and slender cells around TEs that may be TE precursor cells. The TED3 promoter was not significantly activated by wounding. This pattern of expression differed clearly from that of other vascular tissue-related genes such as PAL, 4CL, and GRP1.8. The nature of TED3 promoter enables us to use it to monitor TE differentiation in tissue and to introduce foreign genes preferentially into immature TE.